Marijuana is the most widespread illegal drug of abuse in Western societies. Its main active ingredient, delta-9-tetrahydrocannabinol, acts by binding to specific membrane receptors called cannabinoid receptors. Activation of these receptors exerts intense effects in humans, suggesting that endogenous cannabinoid (endocannabinoid) substances may contribute in important ways to brain functions such as cognition, mood and pain sensation. Several endocannabinoid substances have been identified, including anandamide and 2-arachidonylglycerol (2-AG). Anandamide and 2-AG are released from neuronal and non-neuronal cells and activate cannabinoid receptors with high affinity. After release, anandamide and 2-AG undergo a rapid inactivation process, which may play an important role in terminating their biological actions. They may be taken up by cells via high-affinity transport system(s) and then broken down by distinct enzymatic activities: anandamide by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). In initial studies, we have molecularly cloned a cDNA encoding for rat brain MGL and provided evidence that this enzyme may serve an important function in 2-AG inactivation. Based on these results, we propose to develop novel chemical probes that act as substrates or inhibitors for brain MGL. The first aim of the proposed research is to define the structural requirements involved in the recognition and hydrolysis of 2-AG by MGL, and develop a pharmacophore profile for MGL inhibition. In initial experiments we have molecularly cloned a cDNA encoding for rat brain MGL and developed an adenovirus-mediated MGL over-expression system in mammalian HeLa cells. Using this system, we will explore the structure-activity relationships of 2-AG hydrolysis by rat brain MGL. Specifically, we will design and synthesize novel 2-AG analogs and test them for their ability to serve as substrates or inhibitors for MGL and enhance 2-AG signaling in intact cells. The second aim of the proposed research is to develop chemically reactive 2-AG analogs, which may serve as covalent MGL ligands. Covalent, radioactively labeled ligands for MGL may be useful tools in molecular studies of this enzyme. We will design and synthesize potential covalent ligands for MGL based on the structure of 2-AG, and test them for their ability to interact irreversibly with MGL. These studies will set the stage for the elaboration of potent and selective inhibitors that target either 2-AG or anandamide deactivation and may open novel therapeutic avenues for the treatment of neuropsychiatric and substance abuse disorders. [unreadable] [unreadable]